Project Summary The cell cycle of protozoan parasite Toxoplasma gondii, an important opportunistic pathogen in HIV-AIDS patients, is fundamentally distinct from that of typical eukaryotes. Replication in the actively growing tachyzoites in acute infection and within bradyzoites associated with chronic infection occurs by an internal budding process termed endodyogeny. With endodyogeny, each mother parasite produces 2 daughters per cycle. In contrast, the sexual cycle of the parasite, a stage restricted to the feline gut, occurs by a hybrid of schizogony and endopolygeny which are typically associated with the malaria parasite and members of the genus Sarcocystis respectively. These cell cycle architectures are associated with the generation of multiple progeny per replicative cycle. We recently characterized a cell cycle regulated deubiquitnase TgOTUD3A (TgGT1_258780) the targeted disruption of which relaxed the restriction of tachyzoites replication to endodyogeny (2 progeny per cycle) to exhibit characteristics of both schizogony and endopolygeny-often within the same clonal vacuole. These mutant parasites generate 3,4 or 5 progeny per cycle to define the multi-daughter phenotype. In addition, over 70% of TgOTUD3A tachyzoites ectopically express markers associated with bradyzoites and induce the expression of genes typically associated with merozoties, the stage associated with entry into the sexual cycle in cats. Complementation of the TgOTUD3A-KO failed to restore any of these phenotypes, with whole genome sequencing revealing no credible mutations at the genomic level. This suggested that a compensatory adaptation had occurred. Examination of the other TgOTU family members revealed a selective transcriptional upregulation of two closely related family member, TgOTUD1B (TgGT1_237894) and TgOTUD1C (TgGT1_323200). It is likely that TgOTUD1A (TgGT1_207650) is similarly upregulated. This presents the possibility that one or more of the cell cycle and developmental phenotypes attributed to the loss of TgOTUD3A may in fact be due to the upregulation of the Clade D1 TgOTU members. To address this specific question we propose to functionally characterize the Clade D1 TgOTU members and establish the consequence of both their ablation and regulated overexpression in the wild type and TgOTUD3A-KO background. In doing so we expect to gain insights into how these deubiquitinases govern the selection of cell cycle architecture and control aspects of key developmental transitions cementing an emerging association between these fundamental processes.